Parallel flow cytometer using radiofrequency multiplexing

Abstract

An imaging flow cytometry apparatus and method which allows registering multiple locations across a cell, and/or across multiple flow channels, in parallel using radio-frequency-tagged emission (FIRE) coupled with a parallel optical detection scheme toward increasing analysis throughput. An optical source is modulated by multiple RF frequencies to produce an optical interrogation beam having a spatially distributed beat frequency. This beam is directed to one or more focused streams of cells whose responsive fluorescence, in different frequencies, is registered in parallel by an optical detector.

What is claimed is: 
     
       1. A method comprising:
 irradiating a sample comprising particles in a flow cell with a plurality of independently controlled frequency shifted beams of light; and 
 detecting light from the irradiated particles in the sample with a photodetector. 
 
     
     
       2. The method according to  claim 1 , wherein the method comprises irradiating a frequency shifter component with a laser to generate a local oscillator beam and plurality of radiofrequency-shifted beams of light. 
     
     
       3. The method according to  claim 2 , wherein the frequency shifter component comprises a radiofrequency comb generator configured to generate a plurality of radiofrequency outputs. 
     
     
       4. The method according to  claim 2 , wherein the frequency shifter component comprises one or more of an acousto-optic deflector (AOD) and an acousto-optic frequency shifter (AOFS). 
     
     
       5. The method according to  claim 4 , wherein the frequency shifter component comprises an acousto-optic deflector (AOD). 
     
     
       6. The method according to  claim 1 , wherein the method comprises optically combining the plurality of independently controlled frequency shifted beams of light to generate an optical interrogation beam. 
     
     
       7. The method according to  claim 1 , wherein the plurality of independently controlled frequency shifted beams of light are spatially distributed. 
     
     
       8. The method according to  claim 2 , wherein the method comprises detecting a plurality of beat frequencies from particles propagating through a flow stream in the flow cell, wherein each beat frequency is a frequency difference between the local oscillator beam and each radiofrequency-shifted beam. 
     
     
       9. The method according to  claim 1 , wherein the photodetector comprises a photomultiplier tube (PMT). 
     
     
       10. The method according to  claim 1 , wherein the method further comprises:
 generating one or more waveforms from the detected light; 
 applying a transform to the one or more generated waveforms; and 
 determining a property of the particles based on the one or more transformed waveforms. 
 
     
     
       11. The method according to  claim 10 , wherein the method comprises separating different colors of fluorescence from the irradiated particles in the sample. 
     
     
       12. The method according to  claim 11 , wherein the separated colors of fluorescence are associated with different properties of the particles. 
     
     
       13. The method according to  claim 10 , wherein the method comprises generating the one or more waveforms by digitizing data signals from the photodetector. 
     
     
       14. The method according to  claim 13 , wherein the data signals from the photodetector are digitized using an analog-to-digital converter (ADC), a digitizing oscilloscope or a multi-channel lock-in amplifier. 
     
     
       15. The method according to  claim 14 , wherein the data signals from the photodetector are digitized using an analog-to-digital converter (ADC). 
     
     
       16. The method according to  claim 1 , wherein the particles comprise cells or components of cells. 
     
     
       17. The method according to  claim 1 , wherein the photodetector is configured to simultaneously detect fluorescence from particles at a first modulation frequency and at a second modulation frequency.